Staining nucleic acids is an essential tool in the manipulation of genetic material for any detection or quantitation purpose. The natural bases of DNA are not useful as fluorescent probes because of their extremely low quantum yields, and thus the use of extrinsic probes is necessary.
A common methodology for a sensitive detection of DNA and RNA is the application of fluorescent dyes. The use of these dyes is an attractive alternative to radioactive oligonucleotide labels that require special laboratory facilities and constant precautions to avoid high radiation levels. Currently marketed dyes for nucleic acids staining include the following types of fluorescent markers: (i) intercalating dyes that are incorporated non-covalently to nucleic acids (e.g. cyanine dyes); (ii) minor groove-binding dyes; (iii) large hydrophobic fluorescent dyes (e.g. fluorescein, rhodamine) that are incorporated covalently to nucleotide (oligonucleotide) positions that do not interfere with base pairing (e.g. at the uracil C5, or at the 3′/5′ ends of an oligonucleotide); and (iv) secondary detection method to amplify the signal using a dye or enzyme-labeled streptavidin to detect a biotinylated probe.
These dyes are used in various techniques for detecting genetic material in DNA arrays, gels, in virus particles, and in cells, by fluorescence microscopy or in electrophoresis gels visualized by epi-illumination. Although the above-mentioned fluorescent dyes are highly useful in the field of nucleic acids detection and quantitation, they suffer from many limitations regarding the preparation and application of nucleic acid probes.
Limitations regarding the preparation of nucleic acid probes include: (i) covalent/non-covalent labeling of (oligo)nucleotides by fluorescent dyes require additional experimental procedures involving the use of unique reagents and kits, prior to nucleic acid detection; (ii) various dyes (e.g. ethidium bromide) are chemical hazards; they are potent mutagens and must be handled with extreme care; (iii) various dyes are poorly soluble in water or in phosphate-buffered saline; (iv) in several labeling procedures, nucleotides are labeled by various fluorophores, and then enzymatically incorporated into RNA or DNA probes. In such chemical reactions, several differently labeled conjugates can be produced and hamper the fluorimetric analysis; (v) if measurements are done in solution, the unreacted dye, that has its own fluorescence, may complicate the spectroscopic analysis; (vi) a large hydrophobic dye attached to a nucleotide alters the efficiency of enzymatic incorporation. Thus, samples prepared from labeled nucleotides may have different levels of labeling, making it difficult to compare levels of hybridization between samples; (vii) labeling an oligonucleotide without enzymatic incorporation, e.g., by forming a coordination complex between the nucleic acid and Pt-containing label, results in the labeling of only one dye molecule per 12-20 bases and this labeling ratio may not be sufficient; (viii) a two-step protocol involving first the incorporation of a slightly modified nucleotide into nucleic acid, followed by covalent binding of fluorescent dyes, also suffers the limitation of a relatively small number of dye molecules that can be incorporated (1 dye molecule per 12-20 bases); and (ix) variation of fluorescence yield with degree of dye conjugation to the nucleic acid probe can significantly reduce the reliability of quantitative measures of hybridization-based assays.
Other limitations regarding the application of nucleic acid probes include: (i) the dye may stain other biopolymers; (ii) the use of those stains on gels may result in background fluorescence; and (iii) the intercalating dyes cannot distinguish between RNA and DNA.
An alternative approach to nucleobases staining has been proposed for improving the fluorescence of nucleobases by extension of the natural fluorophore. Adenine has poor fluorescence properties (Callis, 1983). However, bridging the adenine N1,N6-positions by an etheno moiety, such as to obtain N1,N6-etheno-adenosine of formula 1 (see appendix A herein, Y is OH), improves the fluorescence of the parent adenine system (e.g., for 1, λmax 415 nm, φ0.6) (Secrist et al., 1972; Thomas and Leonard, 1976). Over the past three decades, N1,N6-etheno-(2′-deoxy)adenosine (compounds of formula 1, wherein Y is H or OH; ε-d-A/ε-A), as well as other ε-nucleobases such as N3,N4-etheno-(2′-deoxy)cytidine (compounds of formula 2 in Appendix A, wherein Y is H or OH; ε-d-C/ε-C), and N2,N3-etheno-(2′-deoxy)guanosine (compounds of formula 3 in Appendix A, wherein Y is H or oH; ε-d-G/ε-G), have been extensively studied as fluorescent nucleos(t)ide probes. All these probes bear an etheno bridge that represents minimal perturbation to the natural system (Leonard, 1984; Leonard, 1992).
N1,N6-Etheno adenine nucleotides are commonly applied as fluorescent probes for various biochemical studies, such as: structure and function of nucleic acids, protein visualization, enzymatic studies, investigation of nucleotide binding-site, conformational analysis of nucleotides, and pharmacology of nucleosides/nucleotides.
Despite the improved fluorescence properties of 1, as compared to adenosine, its application as a fluorescent probe is limited due to the structural difference between 1 and the natural nucleoside. Thus, N1,N6-ε-A nucleo(s)tides can not be applied to biochemical systems requiring H-bonding based molecular recognition. Specifically, N1 and N6-nitrogens are engaged in an imidazole ring, resulting in the loss of the adenine natural H-bonding capability. Consequently, there is reduction or loss of molecular recognition of these probes by target proteins or nucleic acids.
So far no fluorescent analogue of adenosine (or the corresponding nucleotides) having free N1,N6-positions has been investigated nor disclosed.